Azole dinucleotide compounds and methods for their production

ABSTRACT

A class of novel azole dinucleotide compounds and methods for their production are provided. Compounds of the class typically have pharmacological properties and are well tolerated, being useful, for example, in treating leukemias in warm blooded animals.

TECHNICAL FIELD

This invention is directed to novel azole carboxamide dinucleotidecompounds of adenosine diphosphoribose having pharmacological activity,especially antiviral and antitumor activity, and to methods for theirproduction.

BACKGROUND OF THE INVENTION

ADP-Ribosylation can be defined as the post synthetic modification ofprotein by the covalent attachment of the ADP-ribose moiety ofnicotinamide-adenosine dinucleotide, abbreviated NAD⁺. TheADP-ribosylation of nuclear proteins has recently been reviewed by M. R.Purnell, P. R. Stone, and W. J. D. Whish (Biochem. Soc. Trans., 8, 215,1980).

The enzyme responsible for ADP-ribosylation is poly (ADP-ribose)synthetase. The ADP-ribose moiety of NAD⁺ is cleaved at thenicotinamide-ribose bond and transferred to a protein or a protein-boundADP-ribose molecule to give a protein bound monomer of ADP-ribose or apolymer of ADP-ribose bound covalently to specific protein. This polymermay be enzymatically degraded by an enzyme poly (ADP-ribose)glycohydrolase, which hydrolyzes the pyrophosphate bonds.

In 1958 it was suggested by R. K. Morton (Nature, 181 540, 1958) thatNAD⁺ played a key role in the regulation of cellular proliferation.Hasagawa and coworkers (S. Hasagawa, S. Fujimura, Y. Shimizu and T.Sugimura: Biochem. Biophys. Acta., 149, 369, 1967) pointed out that poly(ADP-ribose) synthetase modified nuclear proteins. In 1976 Rechsteinerand coworkers (M. Rechsteiner, D. Hillyard, and B. M. Olivera: Nature,259, 695, 1976) showed that the cellular half-life of NAD⁺ is one hour,which suggests a high turnover of NAD⁺ due to the utilization by poly(ADP-ribose) synthetase. Thus, more adenine leaves NAD than enters DNA.Caplan and Rosenberg (A. I. Caplan and M. J. Rosenberg: Proc. Nat'l.Acad. Sci., USA, 72, 1852, 1975) were the first to suggest aninvolvement of ADP-ribosylation in cellular differentiation which hassince been supported by other workers (M. R. Purnell, P. R. Stone, andW. J. D. CWhish: Biochem. Soc. Trans., 8, 215, 1980). Berger andcoworkers (N. A. Berger, J. W. Adams, G. W. Sikorski, S. J. Petzold, andW. T. Shearer: J. Clin. Invest., 62, 111, 1978) have shown that chroniclymphatic leukemic cells isolated from patients were higher in poly(ADPR) synthesis than normal cells. It has recently been shown that inEhrlich ascites cells the histone H1 is ADP ribosylated mainly in theC-terminal fragment (H. C. Braeuer, P. Adamietz, U. Nellessen, and H.Hilz: Eur. J. Biochem., 114, 63, 1981). Kidwell (W. R. Kidwell: J.Biochem., 77, 6, 1975) has postulated that poly ADPR formation acts as atrigger for the cell cycle. NAD⁺ levels are known to be lower inmalignant cells than in normal cells (L. S. Jedeiken and S. Weinhouse:J. Biol. Chem., 213, 271, 1955). This may be due to the greaterutilization of NAD⁺ for poly ADPR synthesis in tumor cells.

Novikoff hepatoma cells have been reported to have twice the poly ADPRsynthetase of normal liver cells. (L. Burzio and S. S. Koide: FEBSLetters, 20, 29, 1972). Poly ADPR synthetase activity has been shown tobe 20 times higher in leukemic lymphoblasts as compared to unstimulatednormal lymphocytes (A. R. Lehman, S. Kirk-Bell, S. Shall, and W. J. D.Whish: Exp. Cell Res., 83, 63, 1974). Increased cellular proliferationhas clearly been demonstrated to correlate with increased activity ofpoly ADPR synthetase (For a review see H. Hilz and P. Stone: Rev.Physiol. Biochem. Pharmacol., 76, 1, 1976). Formycin B inhibits cellularproliferation in L-5178Y mouse leukemia cells and it has been postulatedby Muller that its cytostatic action is due to inhibition of poly ADPRformation (W. E. G. Muller and R. K. Zahn: Experientia, 31, 1014, 1975).

Thus we hypothesized that certain novel dinucleotides could be preparedwhich might act as substrate analogs that might bind to poly ADPRsynthetase but could not be utilized by this enzyme, thus resulting inan inhibition of ADP-ribosylation and in controlling rapid cellularproliferation, which would have a direct application in the treatment ofcancer.

Similarly, certain viral regulated ADP-ribosylation processes necessaryfor viral propagation might also be selectively inhibited by the noveldinucleotides which by inhibition of viral replication could be usefulin the treatment of various viral infections.

SUMMARY AND DETAILED DESCRIPTION

The concept of the present invention is to replace the nicotinamidemoiety of NAD⁺ with a unique heterocycle, herein designated as R,containing a carboxamide function which carries no charge at the site ofβ-ribosyl attachment. It is known that the pyridine carboxamide functionis a good leaving group due to the charge at the pyridine nitrogen. Thusa nucleophilic protein function may become ADP-ribosylated byADP-ribosyl synthetase due to a Walden inversion at C₁ via displacementof the nicotinamide group. To inhibit this process one can remove thecharge of the heterocycle at C₁ by:

(1) Attachment of the heterocyclic carboxamide R, via an unchargednitrogen by utilizing a 5-membered ring such as the1,2,4-triazole-3-carboxamide, or

(2) Attachment of R through a carbon-carbon bond such as in the1,3-thiazole-4-carboxamide or the 1,3-selenazole-4-carboxamide.

Since the new and novel dinucleotides, designated hereinafter asStructure I, carry no formal charge in the heterocycle containing thecarboxamide function, these hitherto unknown dinucleotides should beable to penetrate cellular membranes more readily than NAD⁺ and asADP-ribosylation inhibitors could have direct application to thechemotherapy of cancer and the treatment of viral infections. It isimportant that the dinucleotides not inhibit the biochemicaloxidation-reduction reactions of NAD⁺ and its reduced form abbreviatedas NADH since this would result in high host toxicity. Thus, the noveldinucleotide cannot readily accept a hydride ion on the atom adjacent tothe carboxamide function as in NAD⁺. Therefore, these noveldinucleotides specifically provided herein are selected such that theydo not interfere with the oxidation-reduction metabolic pathways of NAD⁺-NADH.

The present invention thus relates to a class of novel dinucleotidecompounds and methods for their production, which compounds areazolecarboxamide compounds of adenosine diphosphoribose (ADP-R) of thestructure I wherein R is a heterocycle selected from4-carbamoyl-1,3-thiazol-2-yl (a), 4-carbamoyl-1,3-selenazol-2-yl (b),and 3-carbamoyl-1,2,4-triazol-1-yl (c); and physiologically acceptablesalts of the azolecarboxamide compounds (I): ##STR1##

Preferred compounds for the purposes of the invention are the following:

P¹ -(Adenosine-5'), P²[(2-β-D-ribofuranosylselenazolecarboxamide)-5'-]pyrophosphate(Selenazole-4-carboxamide Adenosine Dinucleotide), P¹ -(Adenosine-5'),P²-[(1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide)-5'-]pyrophosphate(1,2,4-Triazole-3-carboxamide Adenosine Dinucleoside) and P¹-(Adenosine-5'), P²[(2-β-D-ribofuranosylthiazole-4-carboxamide)-5'-]pyrophosphate(Thiazole-4-carboxamide Adenosine Dinucleotide).

As a general synthetic procedure, nucleotide anhydrides may be preparedby an ion exchange reaction as reviewed by H. G. Khorana ("Some RecentDevelopments in the Chemistry of Phosphate Esters of BiologicalInterest," Wiley, New York, 1961), A. M. Michelson ("Synthesis ofNucleotide Anhydrides by Anion Exchange," Biochim. Biophys. Acta., 91,1-13, 1964), and K. H. Schiet ("Nucleotide Analogs," John Wiley & Sons,New York, 1980). This approach requires that the azolecarboxamide thatis to replace nicotinamide in nicotinamide adenosine dinucleotide (NAD⁺)be converted to its ribotide (phosphoribosylazolecarboxamide) andcondensed with adenosine 5'-monophosphate in base such as pyridine. Avariety of condensing agents (nonenzymatic catalysts) are available forthis synthetic reaction. These mainly serve to activate anoxygen-phosphorus bond such that nucleophilic attack (phosphate-oxygenanion) on the phosphorus atom is facilitated. The condensing agent maybe added to a suspension or solution of the requisite azolecarboxamideribotide and adenosine 5'-monophosphate (A) or the activated nucleotide[either AMP-O-condensing agent (B) or azolecarboxamide-ribose-PO₂-condensing agent (C)] may be preformed and then treated with the other5'-mononucleotide. In this modification the 5'-mononucleotide may besolubilized and activated for nucleophilic attack by the formation oftrialkylammonium or tetraalkylammonium salts (hindered amines): ##STR2##

The nucleotide starting materials may be activated according to theinvention in any suitable way. For example, the nucleotides may beactivated for reaction in situ by condensing agents such asN,N'-dialkylcarbodiimide (DCC) or conversion into isolable, activatedphosphates such as triester pyrophosphates [e.g., P'-diphenyl, P²-(adenosine-5'-)pyrophosphates], phosphoramidates (e.g., adenosine5'-phosphoramidates, -morpholidates, and imidazolidates). The activatedphosphate derived from adenosine 5'-monophosphate is the most practicalroute due to its commercial availability; however, the reverse process(activated phosphate of azolecarboxamide ribotide) is also operationaland adds versatility to this process. The azolecarboxamide adenosinedinucleotide final products can be isolated in any suitable way. Forexample, they conveniently are isolated in pure form by gradient elutionfrom anionic ion exchange chromatography (e.g., AG 1 or 2 and DEAEcellulose).

The compounds of the invention having Structure I are useful antiviraland antitumor agents. Thus, the compounds are typically active, directlyor indirectly against viruses such as the DNA vaccinia virus (VV) andthe RNA vesicular stomatitis virus (VSV) and leukemias such as L1210 andP388 and solid tumors such as the Lewis lung carcinoma as determined bystandard culture assay.

For purposes of providing useful dosage forms, the dinucleotides Isingly or in combination are appropriately mixed with a suitablepharmaceutical carrier which may be sterilized water or physiologicalsaline, i.e., pH and salt adjusted solutions suitable for topical,intravenous, intramuscular, or other routes of administration.

Preferredly, the dinucleotides I of the invention are constituted as asolute in an appropriate pharmaceutical carrier. Alternatively, however,suspensions, emulsions, and other formulations of the compounds of theinvention could be used where indicated. The pharmaceutical carrier, inaddition to having a solubilizing or suspending agent therein, mightalso include suitable dilutants, buffers, surface active agents, andother similar agents as are typically used in pharmaceutical carriers.The total composition of the pharmaceutical carrier, however, is chosento be compatible with the site of delivery and with the concentration ofthe active ingredient.

Each compound of the invention is suitably constituted with thepharmaceutical carrier in a concentration of at least 0.1 percent byweight of the total composition. Preferredly, it would be present in thepharmaceutical carrier at a concentration of about 10% to about 90% byweight of the total composition.

Effective amounts of the dinucleotide, or other compounds of theinvention, typically would range from about 2.5 milligrams per kilogram(mg/kg) of the total body weight of the treated warm blooded animal toabout 200 mg/kg per day. Preferredly, the range would be from 12.5 mg/kgto about 100 mg/kg. An even more preferred range would be from about 15mg/kg to about 50 mg/kg. As with other factors noted above, the amountof compound utilized in treating an afflicted animal would take intoaccount parameters such as the type of virus or tumor, the virus ortumor site, the form of administering the compound, and the physicalsize and condition of the host. In any event, the actual amount shouldbe sufficient to provide a chemotherapeutically effective amount of theagent to the host in a convenient volume.

A composition used for inhibiting malignant tumors in warm bloodedanimals might be suitably prepared by constituting the dinucleotides ofthe invention in a pharmacologically compatible solvent followed bysterilization and packaging in appropriate sealable vials at a knownconcentration. Appropriate doses of the compound are then withdrawn fromthe vial and administered by injection to the host.

The invention is illustrated by the following examples.

EXAMPLE 1

P¹ -(Adenosine-5'), P²[(1-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide)-5'-]pyrophosphate(1,2,4-Triazole-3-carboxamide Adenosine Dinucleotide)

To a solution of adenosine 5'-monophosphate (free acid from SigmaChemical Company , 2.0 g, 5.8 mmol) and1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide 5'-phosphate (U.S. Pat.No. 3,651,045, 0.97 g, 3 mmol) in water (50 ml, initial warming andstirring is necessary to obtain a clear solution) were added anhydrouspyridine (325 ml) and N,N¹ -dicyclohexylcarbodiimide (18 ml). Thereaction mixture was stirred at 0° C. for 24 hours, and then filtered toremove the precipitated dicyclohexylurea. To the filtrate was added asecond portion of DCC (18 ml) and stirring at 0° C. for 24 hours wascontinued. Precipitated dicyclohexylurea was filtered. This process wasrepeated three more times so that a total of 90 ml of DCC were used.Finally, the filtrate was poured into water (2 l) and stirred at roomtemperature for two hours. The solution was filtered, and the filtratewas washed with chloroform (3×350 ml) and the aqueous solution wasconcentrated under reduced pressure to about 50 ml. The pH of thesolution was adjusted to six and applied to a column (20 cm×6 cm) ofDowex-2 resin (formate form). The column was washed with water (4 l) toremove inorganic salts, followed by a gradient elution (water to 0.2 Nformic acid, 1 l each).

The novel dinucleotide was eluted first, followed by adenosine5'-monophosphate and then P¹ P² -diadenosine 5'-pyrophosphate. Thehomogeneous fractions containing the novel nucleotide were combined andconcentrated under reduced pressure (<30° C.) to about 100 ml, frozen,and lyophilized to provide the title product as a white amorphous solid(0.8 g); UV λ max (pH 1) 256 nm (ε 23,800), λ max (pH 7) 258 (25,100), λmax (pH 11) 258 (25,800); 1_(H) NMR (Me₂ SO-d₆)δ 8.50 (s, 1, C₅ H fromtrizole), 8.30 and 8.20 (s, 1, C₂ H and C₈ H from AMP), 7.73 (bs, 4, NH₂and CONH₂, exchanges with D₂ O), 6.0 (d, J=5 Hz) and other sugarprotons.

EXAMPLE 2

P¹ -(Adenosine-5'-), P²-[(2-β-D-ribofuranosylthiazole-4-carboxamide)-5'-]pyrophosphate(Thiazole-4-carboxamide Adenosine Dinucleotide).

2-β-D-Ribofuranosylthiazole-4-carboxamide. Ethyl2-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-thiazole-4-carboxamide wasutilized as prepared in Srivastava et al., J. Med. Chem., 1977, 20, No.2, 256, herein incorporated by reference. A concentrated solution ofethyl 2-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)thiazole-4-carboxamide(5.0 g, 8.31 mmol) in methanol (15 ml) was stirred with methanolicammonia (saturated at 0° C., 100 ml) in a pressure bottle at roomtemperature for two days. The solvent was evaporated and the residue waschromatographed through a column (2.5×35 cm) of silica gel (100 g)packed in ethyl acetate. Elution of the column with a solvent system(ethyl acetate-1-propanol-water, 4:1:2; v/v; top layer) removed thefast-moving methyl benzoate and benzamide. The slower moving, major, UVand sugar-positive fractions were collected and the solvent wasevaporated in vacuo. The residue (syrup), thus obtained, was readilycrystallized from ethanol-ethyl acetate to provide 1.6 g (74%) of pureproduct; mp 144°-145° C.; [α]_(D) ²⁵ 14.3° (c 1, DMF); UV λ max (pH 1)237 nm (8640); UVλ max (pH 11) 238 (8100); ¹ H NMR (Me₂ SO-d₆)δ 7.5 7.8(bs, 2, CONH₂)δ4.99 (d, 1, J=5 Hz, H₁ '), 8.25 (s, 1, H₅).

2-(5-O-Phosphono-β-D-ribofuranosyl)thiazole-4-carboxamide(2-β-D-Ribofuranosylthiazole-4-Carboxamide 5'-Phosphate). Water (151 mg,8.4 mmol) was added carefully to a solution (maintained at 0° C. withstirring) of freshly distilled phosphoryl chloride (2.0 g, 13.2 mmol),pyridine (1.21 g, 14.4 mmol) and acetonitrile (2.3 g, 56.7 mmol).2-β-D-Ribofuranosylthiazole-4-carboxamide (powdered and dried over P₂O₅, 800 mg, 3.0 mmol) was added to the solution and the reaction mixturewas stirred continuously for four hours at 0° C. The reaction mixturewas poured into ice water (ca. 50 ml) and the pH was adjusted to 2.0with 2 N sodium hydroxide. The solution was applied to a column ofactivated charcoal (20 g), and the column was washed thoroughly withwater until the eluate was salt-free. The column was eluted with asolution ethanol-water-concentrated ammonium hydroxide (10:10:1) and 25ml fractions were collected. The fractions containing pure (tlc, silicagel, acetonitrile-0.1 N ammonium chloride (7:3)) nucleotide werecollected and evaporated to dryness under vacuum. The anhydrous residuewas dissolved in water and passed through a column of Dowex 50 W-X8(20-50 mesh, H⁺ form, 15 ml). The column was washed with water and thefraction containing the nucleotide was collected. The solution wasconcentrated to a small volume (5 ml), adjusted to pH 8 with 1 N sodiumhydroxide solution and placed on a column of Bio-Rad AG 1×8 (formateform, 50-100 mesh, 20 ml). The column was first washed with water (100ml) and then with a gradient of 0.2 M to 0.5 M formic acid (300 mleach).

The product appeared after ca. 375 ml of gradient had passed through thecolumn. The pure fractions were pooled and evaporated under reducedpressure (<30° C.) to a small volume. Addition of ethanol (35 ml)provided the desired nucleotide as the free acid as a white powder (ca.500 mg) after washing successively with ethanol and ether and drying at50° for five hours.

P¹ -(Adenosine-5'-)P²-[(2-β-D-ribofuranosylthiazole-4-carboxamide)-5'-]pyrophosphate(Thiazole-4-carboxamide Adenosine Dinucleotide). The title dinucleotideis prepared by condensing 2-β-D-ribofuranosylthiazole-4-carboxamide5'-phosphate (free acid, 1.02 g, 3 mmol) with adenosine 5'-phosphate(Sigma Chemical Company, 2.0 g, 5.8 mmol) in pyridine-water at 0° C. asdescribed in EXAMPLE 1. The crude material was isolated and purified byion exchange chromatography (Dowex-2, formate form) as described inEXAMPLE 1. The novel thiazolecarboximide adenosine dinucleotide wasobtained as a white powder (800 mg); UV λ max (pH 1) 256 nm (ε 10,600),λ max (pH 7) 257 (11,800), λ max pH 11) 257 (12,200):¹ H' NMR (Me₂SO-d₆) δ 6.0 (d, 1, J=5 Hz, H₁ '), 8.3 (s, 1, C₅ H), 8.5 (s, 1, C₈ H),8.7 (s, 1, C₂ H); ir (KBr) 1680 cm⁻¹ (C═O).

EXAMPLE 3

P¹ -(Adenosine-5'-), P²-[(2-β-D-ribofuranosylselenazole-4-carboxamide)-5'-]pyrophosphate(Selenazole-4-carboxamide Adenosine Dinucleotide).

Reaction of 2,5-Anhydro-3,4,6-Tri-O-Benzoyl-D-Allonselenocarboxamidewith Ethyl Bromopyruvate and the Synthesis of Ethyl2-(2,3,5-Tri-O-Benzoyl-D-Ribofuranosyl)selenazole-4-Carboxylates. Asolution of 2,5-anhydro-3,4,6-tri-O-benzoyl-D-allonselenocarboxamide(5.5 g, 10 mmol) in acetonitrile (60 ml) was cooled in ice.Ethylbromopyruvate (3.0 g) in acetonitrile (20 ml) was added dropwise(ten minutes). The ice bath was removed and the reaction mixture wasstirred at room temperature for one hour. The solvent was evaporated invacuo and the residue was triturated with a saturated sodium bicarbonatesolution (100 ml) and extracted with ethyl ether (2×100 ml). Thecombined ether portion was washed with water and dried (MgSO₄). Theether was evaporated in vacuo and the residue (syrup) was passed througha silica gel (300 g) column packed in chloroform. Elution with 5%ethylacetate in chloroform provided subtitle products: namely the fastmoving ethyl2-(2,3,5-tri-O-benzoyl-2-β-D-ribofuranosyl)selenazole-4-carboxylate (2.5g) and the slow moving ethyl2-(2,3,5-tri-O-benzoyl-2-α-D-ribofuranosyl)selenazole-4-carboxylate (1.0g).

2,5-Anhydro-3,4,6-Tri-O-Benzoyl-D-Allonselenocarboxamide. A mixture of2,3,5-tri-O-benzoyl-β-D-ribofuranosylcyanide (10.0 g, 21.2 mmol),4-dimethylaminopyridine (200 mg) and liquid hydrogenselenide condensedunder N₂ atmosphere, 20 ml) was stirred in a sealed bomb at roomtemperature for 20 hours. Hydrogen selenide was allowed to evaporate.The dark colored residue was dissolved in chloroform (200 ml) and washedsuccessively with water (3×50 ml), saturated NaHCO₃ (3×50 ml) followedby water (2×50 ml). The chloroform portion was dried (MgSO₄) andevaporated under vacuum to provide the subtitle product as a foam inalmost quantitative yield. The latter product of analytical purity wasprovided by column chromatography (silica gel, 5% ethyl acetate inchloroform). The product developed a purple color when the silica gelchromatogram of the product was sprayed with a dilute ethanolic solutionof 2,3-dichloronaphthopurinone and exposed to ammonia.

Analysis calculated for C₂₇ H₂₃ NO₇ Se: C, 58.91; H, 4.21; N, 2.54; Se,13.98. Found: C, 58.81; H, 4.29; N, 2.51; Se, 13.74.

2-β-D-Ribofuranosylselenazole-4-carboxamide. Ethyl2-(2-3,5-tri-O-benzoyl-β-D-ribofuranosyl) selenazole-4-carboxylate (3.2g, 5 mmol) was dissolved in methanol (100 ml), cooled and saturated withammonia (0° C.). The solution was stirred in a pressure bottle at roomtemperature for 48 hours. The solvent was evaporated in vacuo and theresidue was extracted with chloroform (25 ml×3). The chloroform portionwas discarded. The residue was absorbed on silica gel (10 g) with theaid of methanol and applied on a silica gel column (2.8×45 cm) packed inethyl acetate. The column was eluted with solvent E (ethyl acetate,n-propanol, H₂ O; 4:1:2; v/v; top layer provides solvent E) and thehomogeneous fractions (Rf≃0.42, silica gel tlc in solvent E) containingthe major product were collected. The solvent was evaporated in vacuoand the title product as a residue was crystallized from 2-propanol:yield 900 mg of the title product (60%) mp 135°-136° C. The residueprovided a second crop (200 mg) with mp 131°-133° C.

Analysis calculated for C₉ H₁₂ N₂ O₅ Se: C, 35.19; H, 3.94; N, 9.12: Se,25.71. Found C, 35.43; H, 3.97; N, 9.03; Se, 25.55.

2-β-D-Ribofuranosylselenazole-4-carboxamide 5'-phosphate. Water (151 mg,8.4 mmol) was added carefully to a solution (maintained at 0° C. bystirring) of phosphoryl chloride (2.0 g, 13.2 mmol), pyridine (1.21 g,14.4 mmol) and acetonitrile (2.3 g, 56.7 mmol).2-β-D-Ribofuranosylselenazole-4-carboxamide (921 mg, 3.0 mmol) was addedto the solution and the mixture was stirred for four hours at 0° C. Aclear solution was obtained which was poured into ice water (50 ml) andthe pH was adjusted to 2.0 with concentrated sodium hydroxide. Thesolution was applied to a column of activated charcoal (30 g), and thecolumn was washed thoroughly with water until the eluate was salt free.The column was eluted with a solution of ethanol-water-concentratedammonium hydroxide (10:10:1) and the fractions (25 ml each) werecollected. The fractions containing the nucleotide product, (tlc, silicagel, acetonitrile - 0.1 N ammonium chloride (7:3)) were collected andevaporated to dryness under vacuum. The anhydrous residual product wasdissolved in water and passed through a column of Dowex 50W-X8 (20-50mesh, H⁺ form, 15 ml). The column was washed with water and the fractioncontaining the nucleotide was collected. The solution was concentratedto a small volume (5 ml), adjusted to pH 8 with 1 N sodium hydroxidesolution, and placed on a column of Bio-Rad AG 1×8 (formate form, 50-100mesh, 20 ml). The column was first washed with water (100 ml) and thenwith a gradient of 0.2 M to 0.5 M formic acid (300 ml each). The productappeared after ca. 375 ml of gradient had passed through the column. Thepure fractions were pooled and evaporated under reduced pressure (<30°C.) to a small volume. Addition of ethanol (35 ml) provided the desirednucleotide as the free acid as a white powder (ca. 500 mg) after washingsuccessively with ethanol and ether and drying at 50° for five hours.

P¹ -(Adenosine-5'-), P²-[(2-β-D-ribofuranosylselenazole-4-carboxamide)-5'-]pyrophosphate(Selenazole-4-carboxamide Adenosine Dinucleotide).2-β-D-Ribofuranosylselenazole-4-carboxamide-5' phosphate (free acid,3.41 g, 10 mmol) is suspended in methanol (70 ml) and tri-n-octylamine(3.39 g, 10 mmol) added. The suspension is then refluxed until a clearsolution is obtained (ca. five minutes). Solvent is removed underreduced pressure and traces of moisture removed from the residue bydissolution in dimethyl formamide followed by evaporation to drynessunder reduced pressure. The residue is dissolved in dioxan (70 ml) andtreated with a solution of P¹ -adenosine-5¹, P² -diphenyl pyrophosphate(5.81 g, 10 mmol) in dioxan (10 ml) and pyridine (23 ml) prepared asdescribed below. To a solution of the tri-n-octylammonium salt (preparedas above) of adenosine 5'-monophosphate (6.98 g, 10 mmol) in dioxane (70ml) is added diphenyl phosphochloridate (3 ml) and tri-n-butylamine (4.5ml) and the clear solution kept at room temperature for two hours underanhydrous conditions. Solvent is then removed under reduced pressure,and ether added to the residue with shaking to precipitate P¹-adenosine-5', P² -diphenyl pyrophosphate. The mixture is kept at 0° C.for 30-60 minutes and then ether is removed by decantation. Dioxan (20ml) is added to the precipitated material and the solution concentratedto a syrup under reduced pressure to remove ether and traces ofmoisture. The residue was dissolved in dioxan and pyridine andvigorously stirred with the tri-n-octylamine salt of2-β-D-ribofuranosylselenazole-4-carboxamide 5'-phosphate (preparedabove) at room temperature for three hours. The solution was mixed withdry ether (600 ml), and the precipitated crude dinucleotide collected bycentrifugation, washed twice with ether, and dried. The crude salt wasdissolved in water and adjusted to pH 6 with 1 N sodium hydroxide andapplied to a column of AG 1×8 ion exchange resin (60×6 cm, formateform). The column was washed with water (3 l) to remove inorganic saltsfollowed by gradient elution (water to 0.2 N formic acid, 1 l each). Thefractions containing the pure selenazolecarboxamide adenosinedinucleotide were pooled and evaporated under reduced pressure (<30° C.)to a small volume. Addition of ethanol (100 ml) precipitated thedinucleotide. This was filtered, washed with ethanol and then ether, anddried at 40° , 0.5 torr for 24 hours to afford the titleselenazole-4-carboxamide adenosine dinucleotide as a white powder (2.0g); UV λ max (pH 1) 255 nm (ε 10,700), λ max (pH 7) 256 (12,000), λ max(pH 11) 256 (12,400); ir (KBr) 1680 cm⁻¹ (CONH₂).

EXAMPLE 4

The dinucleotides of Structure I may be prepared either by procedure A(above), that is by condensation of the appropriate azolecarboxamide5'-ribotide with adenosine 5'-phosphate in the presence ofdicyclohexylcarbodiimide in pyridine-water as described in EXAMPLE 1 orby procedure B (above), which is the reaction of preformedazolecarboxamide 5'-ribotide tri- or tetraalkylammonium salts withpreformed P¹ -adenosine, P² -diphenylpyrophosphate in dioxan-pyridine asdescribed in EXAMPLE 3.

EXAMPLE 5

The dinucleotide free acids as isolated in EXAMPLES 1-3 are eachconverted into amine or alkaline metal salts by passage of an aqueoussolution of the respective dinucleotide free acid through ion exchangeresin (Dowex 50W×8) prepared in the desired counter ion (e.g. Na⁺ Li⁺,K⁺, pyridine, tri-N-butylamine, etc). Thus, using sodium ion exchangeresin the following sodium salts of the invention are obtained: P¹(adenosine-5'-),P²-[(1-B-D-ribofuranosyl-1,2,4-triazole-3-carboxamide)-5'-]pyrophosphate,sodium salt; P¹ (adenosine-5'-),P²-[(2-β-D-ribofuranosylthiazole-4-carboxamide)-5'-]pyrophosphate, sodiumsalt; and P¹ -(adenosine-5'), P²-[(2-β-D-ribofuranosylselenazole-4-carboxamide)-5'-]pyrophosphate,sodium salt.

EXAMPLE 6

Antiviral Evaluation. Inhibition of the virus-induced cytopathic effect(CPE) was used as the indicator of azolecarboxamide adenosinedinucleotide antiviral activity. Inhibition of CPE was observed inAfrican green monkey kidney (Vero, V) cells after infection with herpessimplex virus, type 1 (HSV/1); vaccinia virus (VV); parainfluenza virus,type 3 (PIV/3), and vesicular stomatitis virus (VSV). In theseexperiments, monolayers (18 h) of cells were exposed to 320 TCID₅₀ ofvirus and concentrations of each compound ranging in one-half logdilutions from 1,000 to 1 μg/ml were added within 15 to 30 minutes. Thedegree of CPE inhibition and compound cytotoxicity were observedmicroscopically after 72 hours of incubation at 37° C. and scorednumerically in order to calculate a virus rating (VR) as previouslydescribed by Sidwell, et. al., Appl. Microbiol., 22, 97 (1971).Significance of antiviral activity in terms of VRs has been assigned asfollows: <0.5, slight or no activity, 0.5-0.9, moderate activity, and≧1.0, marked activity. The virus rating (VR) of the dinucleotides havingStructure I range from 0.7 to 1.2. For example, dinucleotide Ic exhibitsa VR of 1.2 against the DNA virus vaccinia (VV) and a VR of 0.7 againstthe RNA virus vesicular stomatitis (VSV).

EXAMPLE 7

Antitumor Evaluation. L1210 cells, a murine leukemia cell line, weregrown in RPMI 1640 supplemented with 5% fetal bovine serum andgentamicin (50 μg/ml).

Drug dilutions were prepared in the appropriate solvent and 20 μl ofeach dilution were added to 24-well Linbro tissue culture platesfollowed by the addition of 2.0 ml of cell suspension containing 3×10⁴cells per ml. Solvent and medium controls were included in each test.After incubation at 37° C. for three days in 5% CO₂, the contents ofeach well were removed and the cells counted in a ZBI Coulter counter.Percent growth was calculated relative to the controls and the levels ofdrug activity were expressed as ID₅₀ in μg/ml using probit paper. Usingthis assay, the dinucleotides of Structure I are active againstleukemias L1210 and P388 and the Lewis lung carcinoma. For example, inthe L1210 leukemia cell culture system Structure Ia has an ID₅₀ of4.2×10⁻⁵ and Structure Ic has an ID₅₀ of 3.5×10⁻⁵.

EXAMPLE 8

The purity and mobility of novel dinucleotides of Structure I wereexamined by high pressure liquid chromatography (HPLC). A Beckman HPLC,model 322, was equipped with a Hidatachi variable wave lengthspectrophotometer (model C-RIA) with peak integrator, and a reversephase column (C-18 ODC Beckman 4.6×25 cm). The dinucleotides ofStructure I and appropriate comparison samples were dissolved in waterand applied to the column by injection. A gradient elution system of 0.1KH₂ PO₄ and 5 mM tetrabutylammonium phosphate buffer (pH 5) cleanlyseparated all peaks. Retention times were for Structure Ia, 13.93minutes; Structure Ib, 14.05 minutes; adenosine 5'-phosphate, 14.22minutes; adenosine 5'-diphosphate, 22.68 minutes; and adenosine5'-triphosphate, 26.08 minutes.

Dinucleotides of Structure I were found by this HPLC instrumentation anddeveloping system to be highly pure. For example, dinucleotide Ia was98.3% pure and dinucleotide Ib was 97.7% pure.

What is desired to claim as our exclusive privilege and property in theinvention as described is the following:

We claim:
 1. Azolecarboxamide adenosine dinucleotide compounds havingthe structural formula: ##STR3## and the pharmaceutically acceptablesalts of the compounds; wherein R is a heterocycle that is3-carbamoyl-1,2,-4-triazol-1-yl or 4-carbamoylselenazol-2-yl.
 2. P¹-(Adenosine-5'-), P²-[(1-B-D-ribofuranosyl-1,2,4-triazole-3-carboxamide)-5'-]pyrophosphateand pharmaceutically acceptable salts thereof.
 3. A compound accordingto claim 2 which is P¹ -(adenosine-5'-), P²-[1-B-D-ribofuranosyl-1,3,4-triazole-3-carboxamide)-5'-[pyrophosphate.4. A compound according to claim 2 which is P¹ -(adenosine-5'-), P²-[(1-B-D-ribofuranosyl-1,2,4-triazole-3-carboxamide)-5'-[pyrophosphate,sodium salt.
 5. A compound according to claim 1 which is P¹-(adenosine-5'-),P²-[(2-β-D-ribofuranosylselenazole-4-carboxamide)-5'-[pyrophosphate andpharmaceutically acceptable salts thereof.
 6. A compound according toclaim 1 which is P¹ -(adenosine-5'-),P²-[(2-β-D-ribofuranosylselenazole-4-carboxamide)-5'-[pyrophosphate.
 7. Acompound according to claim 1 which is P¹ -(adenosine-5'-),P²-[(2-β-D-ribofuranosylselenazole-4-carboxamide)-5'-[pyrophosphate,sodium salt.